Cryptococcus neoformans is an encapsulated budding yeast that causes a life-threatening illness in immunocompromised individuals, such as patients with AIDS, organ transplant recipients, and those treated with immunosuppressive therapies. Although the infection starts in the lung, cryptococcosis commonly presents as meningoencephalitis, which is one of the most common infections of the central nervous system and the leading cause of death in HIV-infected individuals. Hematogenous dissemination of C. neoformans from the lung to the brain is believed to be the most critical step in the progression of cryptococcal meningoencephalitis. How it happens, however, remains poorly understood because of the technical challenge of in vivo studies. C. neoformans is a facultative intracellular pathogen that can survive in phagocytes. Our preliminary study has provided direct evidence that C. neoformans is engulfed in the lung and subsequently disseminate to the blood within phagocytes. As neutrophils are the most abundant phagocytes in the bloodstream and have been shown to be recruited to the infected lung for phagocytosis of the yeast cells, we have developed a novel in vivo phagocytosis model to study the migration of neutrophils containing ingested C. neoformans using intravital microscopy. Based on our preliminary data showing that considerable numbers of neutrophils containing C. neoformans roll/crawl and adhere to brain vasculature after infection, we hypothesize that neutrophils mediate brain dissemination of C. neoformans by nonlytic exocytosis of the fungus and direct cell-to-cell spread of the intracellular fungus from adherent infected neutrophils to endothelial cells in the brain vasculature. We will test the hypothesis in vitro as well as in vivo by addressing the following aims using intravital microscopy and other advanced approaches: (1) Investigate migration of neutrophils harboring ingested C. neoformans from the lung to the brain vasculature; (2) Characterize nonlytic exocytosis of C. neoformans by neutrophils and the BBB crossing of the yeast cells via a direct cell-to-cell spread from adherent infected neutrophils to endothelial cells in the brain vasculature. The proposed studies will provide unique insights into brain dissemination of C. neoformans, facilitating the development of novel prevention and therapeutic strategies.